Implication of self-organizing map, stable isotopes combined with MixSIAR model for accurate nitrogen control in a well-protected reservoir.

Nitrogen pollution and eutrophication in reservoirs is a global environmental geochemical concern. Occasional algal blooms still exist in reservoirs that have undergone pollution treatment. The lack of quantitative evidence of nitrogen sources and fate limits long-term stable ecological safety management. This work applied an approach integrated zonal mapping, stable isotopes (δ18OH2O, δ15Nnitrate, δ18Onitrate, and δ13C-DIC) and a Bayesian isotope model to analyze regional and seasonal differences in the contribution and sources of nitrogen to a well-protected reservoir. The values of δ18Onitrate and the positive relationship between NO3- and δ13C-DIC suggested that nitrification was the primary NO3- production in the rivers. While Denitrification was present at only a few sites. Results of the MixSIAR model coupled the NO3-/Cl- indicator revealed that the domestic sewage contributed high riverine NO3- loading (68.6 ± 10.6 %) in the dry season. In the wet season, the main nitrate sources of upper watershed were ammonia and carbamide fertilizers (47.5 % and 40.3 %). While the domestic sewage was still the major contributor of downstream region (a dense residential area), indicating possible problems with rainwater and sewage drainage networks. The results implied that the colleting and treatment of sewages were the priority in downstream region, and non-point source pollution control and wastewater treatment plant upgrading were essential to control nitrate pollution in the two upstream regions. These findings provide new insights into precise nitrogen pollution traceability and identification of treatment priorities in the sub-region, and promote the management other well-protected watershed in similar need of further nitrogen contamination control.

Subclinical Diabetic Peripheral Vascular Disease and Epidemiology Using Logistic Regression Mathematical Model and Medical Image Registration Algorithm.

The study aims to explore the effect of subclinical diabetic peripheral vascular disease and an epidemiological investigation of colour Doppler ultrasound images based on a logistic regression mathematical model and a medical image registration algorithm. Subclinical diabetes patients were selected as subjects, and after ultrasound colour Doppler ultrasonography of peripheral blood vessels, ultrasound images were taken. The experimental results show that the area under the curve (AUC) predicted by the model was 0.748, the sensitivity was 94.12%, and the specificity was 67.93%. All Δ were smaller than a single pixel. The detection rate of colour Doppler ultrasonography was 82.6%, which was significantly better than that of clinical examination (P < 0.01). The age, course of disease, SBP, low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and triglyceride (TG) of the peripheral vascular disease group were significantly different from those of the no peripheral vascular disease group (P < 0.05). The incidence of peripheral vascular diseases and nonperipheral vascular diseases in male patients was remarkably higher than that in female patients (P < 0.05). Moreover, with the increase of age, the incidence of peripheral vascular disease and nonperipheral vascular disease in diabetic patients showed a trend of gradual increase (P < 0.05). In summary, the mathematical model and registration method have high accuracy for medical image registration of patients with the diabetes epidemic. In addition, the age, course of disease, SBP, LDL-C, TG, and TC of diabetic patients were significantly different from those of normal people, which can provide a reference for the development of later diabetes epidemiology.

Molecular mechanism of miR-34b-5p and RNA binding protein HuR binding to lncRNA OIP5-AS1 in colon cancer cells.

Colon cancer (CC) is a leading cause of cancer-related death. Long non-coding RNA OIP5-AS1 (lncRNA OIP5-AS1) expression pattern has been studied in many cancers. We aimed to identify the mechanism of lncRNA OIP5-AS1 in CC development. OIP5-AS1 expression pattern in CC tissues and cells was detected and the relation between OIP5-AS1 level and CC prognosis was analyzed. The proliferation, migration and invasion of CC cells were detected after silencing or overexpression of OIP5-AS1. Tumor xenograft in nude mice was established to verify the effect of OIP5-AS1 in vivo. The interaction between HuR protein and OIP5-AS1 and the interaction of miR-34b-5p with HuR and OIP5-AS1 were measured. OIP5-AS1 was highly expressed in CC and associated with poor prognosis. Silencing OIP5-AS1 inhibited CC cell malignant behaviors and inhibited the growth rate and tumor weight. In the mechanism, HuR bound to OIP5-AS1 and stabilized OIP5-AS1 expression. Both miR-34-5p and HuR bind to OIP5 and oppositely affect its expression. miR-34b-5p inhibited the proliferation and invasion of CC cells by inhibiting OIP5-AS1 and PI3K/Akt pathway. miR-34b-5p inhibited CC growth by inhibiting OIP5-AS1. Collectively, miR-34b-5p targets HuR and miR-34b-5p binds to OIP5-AS1 with HuR, thus inhibiting OIP5-AS1 and PI3K/Akt pathway and CC progression.

Exosomes miR-22-3p Derived from Mesenchymal Stem Cells Suppress Colorectal Cancer Cell Proliferation and Invasion by Regulating RAP2B and PI3K/AKT Pathway.

OBJECTIVE: Exosomes (exo) which contain proteins, microRNAs (miRNAs), and other bioactive substances can participate in intercellular signal transduction and material transport. Bone marrow mesenchymal stem cells (BMSCs) have a strong ability to produce exosomes. The purpose of this study was to observe the effect of hBMSCs-derived-exo miR-22-3p on proliferation and invasion of colorectal cancer (CRC) cells and to explore its mechanism. METHODS: miR-22-3p and RAS oncogene family (RAP2B) expression was detected using qRT-PCR or Western blotting. Their interaction was confirmed by dual luciferase activity assay. Effects of miR-22-3p on cell proliferation and invasion were evaluated by CCK-8 and Transwell assay, respectively. Exosomes were extracted by the ultracentrifugation and identified through electron microscopy and Western blotting. RESULTS: In CRC tissues and cells, downregulation of miR-22-3p and upregulation of RAP2B were observed. According to the analysis of dual luciferase activity, RAP2B was a target gene of miR-22-3p. In addition, miR-22-3p obviously repressed the cells proliferation and invasion via mediating RAP2B/PI3K/AKT pathway. Coculture experiments indicated that miR-22-3p derived from hBMSCs-exo had inhibition effects on SW480 cell proliferation and invasion. CONCLUSIONS: Collectively, miR-22-3p from hBMSCs-exo might impede CRC progression, which emphasized the potential of hBMSCs-exo-miR-22-3p as CRC treatment in the future.

Low Grip Strength is a Strong Risk Factor of Osteoporosis in Postmenopausal Women.

OBJECTIVE: To investigate the effect of grip strength on bone mineral density (BMD) in postmenopausal women. Low BMD is related to risk of fracture and falling is the strongest factor for fragility fractures. Handgrip strength is a reliable indicator of muscle strength and muscle strength is associated with falling. METHODS: For the present study 120 women were divided into two groups: those ≤65 years and those >65 years. Serum 25 hydroxyvitamin D (25OHD), BMD, and handgrip strength were measured to observe the effect of age on 25OHD, grip strength, and BMD, as well as the effect of 25OHD on grip strength and BMD. The correlation between grip strength and BMD was investigated. RESULTS: In the 120 patients, 25OHD was 24.31 ± 8.29 ng/mL. There were 37 cases with 25OHD <20 ng/mL and 83 cases with 25 OHD ≥20 ng/mL. The patients with 25OHD <20 ng/mL had significantly lower femoral neck BMD, most of them with a T score ≤-2.5 (P < 0.05). BMD measurement showed 66 patients with femoral neck T ≤-2.5, 30 cases with total hip T ≤-2.5 and 90 cases with lumbar BMD T ≤-2.5. The maximum grip strength in the group is 22.28 ± 6.17 kg. There were 38 cases with the maximum grip strength <20 kg and 82 cases with the maximum grip strength ≥20 kg. Patients >65 years had lower 25OHD, lower maximum grip strength, and lower BMD. The osteoporosis risk in postmenopausal women with a maximum grip strength <20 kg and who were >65 years was significantly elevated. CONCLUSION: Handgrip strength and 25OHD decrease with aging in postmenopausal women. The patients with lower 25OHD level had significantly lower BMD of femoral neck. The patients with lower handgrip strength had significantly lower BMD of lumbar spine, femoral neck, and total hip. Grip strength measurement is the simplest muscle strength measurement method. Our study confirmed that low grip strength was correlated with low BMD and was a strong risk factor for osteoporosis in postmenopausal women.

Identification and Genetic Analysis of a Factor IX Gene Intron 3 Mutation in a Hemophilia B Pedigree in China.

OBJECTIVE: Hemophilia B is caused by coagulation defects in the factor IX gene located in Xq27.1 on the X chromosome. A wide range of mutations, showing extensive molecular heterogeneity, have been described in hemophilia B patients. Our study was aimed at genetic analysis and prenatal diagnosis of hemophilia B in order to further elucidate the pathogenesis of the hemophilia B pedigree in China. MATERIALS AND METHODS: Polymerase chain reaction amplification and direct sequencing of all the coding regions was conducted in hemophilia B patients and carriers. Prenatal diagnosis of the proband was conducted at 20 weeks. RESULTS: We identified the novel point mutation 10.389 A>G, located upstream of the intron 3 acceptor site in hemophilia B patients. The fetus of the proband's cousin was identified as a carrier. CONCLUSION: Our identification of a novel mutation in the F9 gene associated with hemophilia B provides novel insight into the pathogenesis of this genetically inherited disorder and also represents the basis of prenatal diagnosis.

SOX9 overexpression plays a potential role in idiopathic congenital talipes equinovarus.

The collagen, type IX, alpha 1 (COL9A1) gene was previously identified as a candidate gene for idiopathic congenital talipes equinovarus (ICTEV), a congenital lower limb deformity in humans. In the present study, increased expression levels of COL9A1 were identified in the abductor hallucis muscle of ICTEV patients compared with those in control samples. The COL9A1 gene is regulated by SRY (sex­determining region Y)­box 9 (SOX9). Immunofluorescence analysis of SOX9 and COL9A1 proteins identified colocalization to the sarcolemma, endomysium and muscle membrane in muscle samples of ICTEV. No mutations in the exons and promoters of SOX9 were detected in blood samples of 84 ICTEV patients by denaturing gradient gel electrophoresis. mRNA and protein expression levels of SOX9 were detected by real­time polymerase chain reaction and western blot analysis, respectively and were found to be significantly higher in ICTEV muscle samples compared with those in control samples. Based on present observations, we hypothesize that overexpression of the SOX9 gene may play a role in the genetic etiology of ICTEV.

[Mechanism of GLI3 gene transcription regulation in idiopathic congenital talipes equinovarus].

OBJECTIVE: To investigate the mechanism of transcription regulation of GLI3 gene in idiopathic congenital talipes equinovarus. METHODS: pGL3-Gli3 luciferase report vectors were constructed, and the activity of Gli3 promoter was explored. A P-Match software was used to analyze the sequence upstream of the transcription start site of rat Gli3 gene, which was subsequently verified with chromatin immunoprecipitation assay (CHIP) and electrophoretic mobility shift assay (EMSA). Expression of the Gli3 gene was analyzed in L6 cells transfected with Hoxd13 small interference RNA(siRNA) and Hoxd13 expression vectors. RESULTS: The 5' region of rat Gli3 gene contains two potential binding sites for the Hoxd13 protein. CHIP and EMSA assays both confirmed that Hoxd13 can directly bind with site 2. As shown in L6 cells, expression of Gli3 may be enhanced with silencing of Hoxd13, whilst exogenous expression of Hoxd13 can down-regulate transcription of Gli3. CONCLUSION: Hoxd13 can directly regulate the expression of Gli3 gene through a Hoxd13 binding site in the limb of rat embryo.

[Possible role of GLI3 gene in the pathogenesis of idiopathic congenital talipes equinovarus].

OBJECTIVE: To investigate the relationship between GLI3 gene and pathogenesis of idiopathic congenital talipes equinovarus (ICTEV). METHOD: Potential mutations in the coding region of GLI3 were detected among 84 patients with ICTEV by denaturing gradient electrophoresis. Expression of GLI3 in the ICTEV patients' disease tissues was assessed by reverse transcription PCR. Following generation of rat model for ICTEV, mRNA and protein levels of GLI3 were evaluated by real-time PCR and immunohistochemistry and Western blotting. RESULTS: No mutation was found in exons 1 - 8 and 13 of GLI3 gene among the 84 ICTEV patients. No expression of GLI3 gene was detected in the flexor hallucis longus of ICTEV patients or normal controls. Expression of Gli3, in terms of both mRNA and protein, was stronger in the hindlimb of ICTEV rat embryos compared with normal controls. CONCLUSION: Mutation in the coding region of GLI3 may not be responsible for the occurrence of ICTEV. However, there may still be connection between abnormal expression of the gene and pathogenesis of ICTEV.

[Expression of COL9A1 gene and its polymorphism in children with idiopathic congenital talipes equinovarus].

OBJECTIVE: COL9A1 gene is located in the susceptibility region of idiopathic congenital talipes equinovarus (ICTEV) (6q12-13). This study aimed to investigate the expression of the COL9A1 gene and the distribution of single nucleotide polymorphism (SNP) of COL9A1 gene in patients with ICTEV and normal controls. METHODS: Immunohistochemistry was used to detect the expression of COL9A1 in 25 children with ICTEV and 5 normal controls. The frequencies of genotypes and allele of two SNPs in COL9A1 gene rs35470562 and rs1135056 were investigated by PCR-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing in 118 patients with ICTEV and 100 normal controls. RESULTS: The COL9A1 protein expression was significantly higher in 22 (88%) out of 25 children with ICTEV than normal controls. There were significant differences in the frequencies of genotypes and allele of rs1135056 in COL9A1 gene between the ICTEV and the control groups: the G allele frequency was higher, the frequency of AA genotype was lower, and the frequencies of AG and GG genotypes were higher in ICTEV patients than those in healthy controls (P<0.05). CONCLUSIONS: COL9A1 protein is highly expressed in patients with ICTEV and rs1135056, which is located in the coding region of COL9A1 gene, may be associated with the pathogenesis of ICTEV.

[The regulation of UTROPHIN expression by EN1].

To investigate possible factors up-regulating the expression of UTROPHIN, potential regulatory elements in the promoter of the human UTROPHIN was predicted by P-match software and verified by EMSA and ChIP. The mechanism of EN1 regulation of the human UTROPHIN expression was evaluated by RNA interference and real-time PCR analyses. Two potential EN1 binding sites in UTROPHIN promoter region were predicted by P-Match software but only the second site was verified to interact directly with EN1 by EMSA and ChIP. The results from RNA interference and real-time PCR showed that the mRNA level of UTROPHIN increased in HeLa cells after EN1 was knockdowned by siRNA. It indicated that EN1 might be a negative regulatory factor for UTROPHIN. Our study suggested that UTROPHIN might be a new target for DMD therapy.

Duchenne or Becker muscular dystrophy: a clinical, genetic and immunohistochemical study in China.

BACKGROUND AND OBJECTIVE: Duchenne and Becker muscular dystrophies are X-linked diseases caused by mutations in the dystrophin gene, which affect approximately 1 in 3,500 and 1 in 18,000 boys, respectively. The aim of this work was to develop a method to assist the diagnosis and classification of the disease. MATERIALS AND METHODS: A large data set of dystrophin mutations was detected in 167 Chinese patients by multiplex ligation-dependent probe amplification and sequencing. Muscle biopsy, immunohistochemistry and STR analysis were also carried out in the patients and carriers. RESULTS: One hundred and three deletions, 23 duplications and two-point mutations. The deletion of one or more exons was detected in 103 (61.7%) patients. The region spanning exons 44-55 was the most frequent deletion. The duplication was identified in 23 (13.8%) patients, which was more common than previously reported. Most duplications were found in exons 2-18. Six out of the 45 muscle biopsies analyzed showed the presence of other muscle diseases. CONCLUSIONS: This study may be important to enable comparisons of mutation type and the most appropriate analytical approach for samples from different geographical areas and ethnicities.

The expression of Gli3, regulated by HOXD13, may play a role in idiopathic congenital talipes equinovarus.

BACKGROUND: Idiopathic congenital talipes equinovarus (ICTEV) is a congenital limb deformity. Based on extended transmission disequilibrium testing, Gli-Kruppel family member 3 (Gli3) has been identified as a candidate gene for ICTEV. Here, we verify the role of Gli3 in ICTEV development. METHODS: Using the rat ICTEV model, we analyzed the differences in Gli3 expression levels between model rats and normal control rats. We used luciferase reporter gene assays and ChIP/EMSA assays to analyze the regulatory elements of Gli3. RESULTS: Gli3 showed higher expression levels in ICTEV model rats compared to controls (P < 0.05). We identified repressor and activator regions in the rat Gli3 promoter. The Gli3 promoter also contains two putative Hoxd13 binding sites. Using EMSA, the Hoxd13 binding site 2 was found to directly interact with Hoxd13 in vitro. ChIP assays of the Hoxd13-Gli3 promoter complex from a developing limb confirmed that endogenous Hoxd13 interacts with this region in vivo. CONCLUSION: Our findings suggest that HoxD13 directly interacts with the promoter of Gli3. The increase of Gli3 expression in ICTEV model animal might result from the low expression of HoxD13.

[Prenatal diagnosis of Duchenne and Becker muscular dystrophy by multiplex ligation-dependent probe amplification].

Duchenne/Becker muscular dystrophy (DMD/BMD) is an X-linked lethal recessive disease caused by mutation in the DMD gene. There is no efficient treatment for this serious and disabling disease. We established a combination method to detect carriers and performed prenatal diagnosis. Using multiplex ligation-dependent probe amplification (MLPA) and linkage analysis of short tandem repeats (STR) methods, 26 prenatal diagnosis were performed for pregnancies at risk of having a DMD/BMD baby through amniocentesis. Seven out of 26 male fetuses were affected and the pregnancies were terminated. Four out of 26 female fetuses were found to be carriers. MLPA can be the method of choice for initial screening of DMD/BMD patients for deletions and duplications mutations. When combined with STR-based analysis, it can improve the rate of DMD/BMD prenatal diagnosis.

[Gene diagnosis for spinal muscular atrophy and its application study].

OBJECTIVE: To establish an effective testing system for gene diagnosis, carrier detection and prenatal diagnosis for spinal muscular atrophy (SMA). METHODS: Twenty-six patients with SMA were directly tested with PCR-RFLP for exon 7 deletion in the SMN1 gene. Carrier detection was carried out with multi-PCR-DHPLC. Amniotic fluid was taken at the middle stage of gestation from pregnant women who had given birth to affected children. RESULTS: Twenty-five out of 26 patients were diagnosed as having SMN1 gene deletion. Fifty-two of their parents were found to be carriers of exon 7 deletion. Eight of 20 fetuses were diagnosed as having SMN1 gene deletion by PCR-RFLP. CONCLUSION: PCR-RFLP and multi-PCR-DHPLC techniques can provide rapid diagnosis for exon 7 deletion detection and carrier detection. PCR-RFLP may also be adapted for prenatal gene diagnosis of exon 7 deletion in SMN1 gene.

Characteristics of dystrophin gene mutations among Chinese patients as revealed by multiplex ligation-dependent probe amplification.

AIMS: To verify whether dystrophin gene mutations among Chinese patients feature different types and frequencies from other populations. METHODS: Multiplex ligation-dependent probe amplification (MLPA) in combination with multiplex PCR (mPCR) and/or short tandem repeat (STR)-based linkage analysis were applied in a large series of Chinese families affected with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). There were a total of 19 cases seeking prenatal diagnosis during their second pregnancies. RESULTS: Of the 59 family trios (51 with DMD and 8 with BMD), 40 were found to have carried various mutations of the dystrophin gene. In addition to deletions and duplications within the mutational hotspots identified by both methods, 10 mutations missed by mPCR were detected by MLPA, among which at least 3 were of rare types. Combined MLPA and linkage analysis also achieved prenatal diagnoses in all of the 19 amniocentesis samples. CONCLUSIONS: Mutations of dystrophin gene among Chinese patients showed a diverse spectrum, with similarity to as well as discrepancies from other populations. For the comprehensive coverage of all exons of the dystrophin gene, MLPA should be the method of choice for initial screening of DMD/BMD patients. When combined with STR-based analysis, it can achieve diagnosis in as much as 70-80% of all referred cases.

[The functions of GLI3 and EN1 genes in idiopathic congenital talipes equinovarus].

To investigate the role of gene Gli3 in idiopathic congenital talipes equinovarus (ICTEV), we constructed the Gli3 luciferase reporter gene expression vectors to analyze the promoter activity of the rat gene Gli3. The regulatory element in the promoter region of the rat Gli3 was predicted using P-Match software and further verified by ChIP experiment. Meanwhile, the correlation between the rat En1 and ICTEV was evaluated by RT-PCR, immunohistochemistry, and Western blotting analyses. The result from P-Match software prediction showed that only one of the three possible En1 binding sites in Gli3 promoter region was interacted directly with En1 in vivo, which was confirmed by ChIP analysis. The results from RT-PCR, immunohistochemistry and Western blotting analyses suggested that En1 was down-regulated in ICTEV model rats compared to the controls. Our results indicated that En1 might be the negative regulatory element in the upstream of Gli3. The low expression level of EN1 in ICTEV could contribute to the up-regulation of GLI3, which led to the genesis of ICTEV.

[Association study between mutations of transcription regulator sequences of COL1A1 gene and idiopathic con-genital talipes equinovarus].

RT-PCR was used to detect the expressions of COL1A1 mRNA in 20 patients with idiopathic congenital talipes equinovarus (ICTEV). The primers were designed by Primer 5 according to sequences of -1 031 bp~ +30 bp and the first intron of COL1A1. PCR-DGGE was used to screen the mutations in COL1A1 gene. Expression of COL1A1 on mRNA levels showed significantly higher in patients with ICTEV than in normal persons (t=12.680, P < 0.05). By DNA sequencing, a -161(T--> C) heterozygous mutation and a+ 274(C-->G) homozygous mutation were detected, and both were new identified mutations. These results indicated that the mutations in transcription regulator sequences of COL1A1 could cause ICTEV.

[Detection of new mutations in the dystrophin gene by denaturing high-performance liquid chromatography].

OBJECTIVE: Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by dystrophin gene mutations; 55%-65% of these pathogenic mutations are large deletion and duplication mutations that can be detected by multiplexed polymerase chain reaction. However, finding the remaining micro-mutations (substitutions, deletions or insertions of one or several nucleotides) cannot be achieved in this way. The aim of the present study was to detect mutations of the dystrophin gene in individuals with Duchenne muscular dystrophy (DMD) by denaturing high-performance liquid chromatography (DHPLC) and to establish a rapid and sensitive screening platform for micro-mutations leading to DMD. METHODS: Twenty patients negative for large deletions in the dystrophin gene by multiplex PCR were selected for further screening by DHPLC and 20 normal male without DMD family history as the control cohort. Dystrophin exons and their flanking sequences were individually amplified by genomic PCR and the amplicons showing abnormal DHPLC profile were directly sequenced to identify the position and the type of the mutations. RESULTS: After screening 68 exons covering the two deletion hotspots and 3'UTR region, four pathogenic mutations, including c.6808_6811del TTAA, c.4959_4960insA, c.8656C > T and c.8608C > T, were found in four DMD patients. Moreover, c.6808_6811del TTAA, c.4959_4960ins and c.8656C > T have not been reported previously. The first two frameshift mutations were predicted to produce premature stop codons, p.Leu2270MetfsX9 and p.Ser1654LysfsX5, respectively. The remaining two were nonsense mutations, leading to p.R2886X and p.R2870X, respectively. CONCLUSION: Three novel and one recurrent dystrophin mutations have been identified in Chinese DMD patients. This study has demonstrated that DHPLC is an effective screening method for micro-mutation associated with DMD.

[Application study on inversion diagnosis of F8 gene in hemophilia A].

OBJECTIVE: To establish an effective method of genetic diagnosis on hemophilia A (HA) by detecting the inversion mutation in intron 22 of F8 gene. METHODS: Intron 22 inversion mutation in F8 gene was detected by using long distance-polymerase chain reaction (LD-PCR) and inversion-PCR (I-PCR) in 31 HA patients. The mothers of HA patients with intron 22 inversion mutation were selected to carrier diagnosis and amniotic fluid of the pregnant women with inversion mutation was collected at intermediate stage of gestation, and used to prenatal genetic diagnosis. RESULTS: Seven patients showed F8 gene inversion mutation in thirty-one patients. Three in four mothers of HA patients with intron 22 inversion mutation were diagnosed as carriers. The prenatal diagnosis result indicated that the fetus conceived in the HA-carrier woman was normal individual. CONCLUSION: The detection of intron 22 inversion mutation by LD-PCR and I-PCR is time-saving, and can be used in prenatal diagnosis on HA.